International Journal of Systematic and Evolutionary Microbiology (2008), 58, 89–92
DOI 10.1099/ijs.0.65118-0
Prevotella amnii sp. nov., isolated from human
amniotic fluid
Paul A. Lawson,1 Edward Moore2 and Enevold Falsen2
Correspondence
1
Paul A. Lawson
2
[email protected]
Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019-0245, USA
Culture Collection, Department of Clinical Bacteriology, University of Göteborg,
SE-413 46 Göteborg, Sweden
Two Gram-negative, anaerobic, non-spore-forming, rod-shaped organisms were isolated from
human amniotic fluid. Based on morphological and biochemical criteria, the strains were
tentatively identified as Bacteroidaceae but they did not appear to correspond to any recognized
species of this family. Comparative 16S rRNA gene sequencing studies showed the strains
were highly related to each other and confirmed their placement in the genus Prevotella, but
sequence divergence values of .4 % with reference Prevotella species demonstrated that the
organisms from human clinical sources represent a novel species. Phylogenetic analysis revealed
the novel organism to be most closely related to Prevotella bivia, an organism frequently
associated with pelvic inflammatory diseases. The major long-chain cellular fatty acids of the novel
species consist of iso-C14 : 0, anteiso-C15 : 0, iso-C15 : 0, C16 : 0, iso-C16 : 0 and iso-3-OH-C17 : 0.
Based on biochemical criteria and phylogenetic considerations, it is proposed that the unknown
isolates from human amniotic fluid be assigned to a new species of the genus Prevotella, as
Prevotella amnii sp. nov. The type strain of Prevotella amnii is CCUG 53648T (5JCM 14753T).
The genus Prevotella includes moderately saccharolytic,
bile-sensitive species formally belonging to the genus
Bacteroides (Shah & Collins, 1990). Many species of
Prevotella have been isolated from human sources often
associated with the oral cavity; indeed of the recently
described species all have been isolated from this location
(Downes et al., 2005; Sakamoto et al., 2004, 2005a, b).
However, a number of species have been isolated from
both healthy and infected tissues of the pelvic region,
including the genitourinary tract, vagina and cervix (Brook
& Frazier, 1997; Fredricks et al., 2005; Hyman et al., 2005;
Mikamo et al., 1999; Puapermpoonsiri et al., 1997). During
a continuing study of Gram-negative anaerobic rods, two
strains were deposited with the Culture Collection of the
University of Göteborg (CCUG), Sweden. Based on the
results of a polyphasic taxonomic study, it is proposed that
the isolates be assigned to the genus Prevotella, as Prevotella
amnii sp. nov.
Isolate CCUG 53648T was recovered from the amniotic
fluid of a 29-year-old female in 1999, the fluid was
described as turbid and ill smelling; CCUG 43050 was
isolated from a 26-year-old female in 2006, again from
amniotic fluid. The unidentified organisms were cultured
on Columbia agar supplemented with 5 % defibrinated
horse blood (Oxoid) at 37 uC, in an atmosphere of 85 %
nitrogen, 10 % carbon dioxide and 5 % hydrogen. The
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain CCUG 53648T is AM422125.
65118 G 2008 IUMS
strains were characterized biochemically by using a
combination of conventional tests as described in the
VPI Anaerobe Laboratory Manual (Holdeman et al., 1977),
and API rapid ID32AN and API ZYM systems according to
the manufacturer’s instructions. Fermentation tests were
performed using pre-reduced, anaerobically sterilized
(PRAS) peptone-yeast (PY)-sugar broth tubes. All biochemical tests were performed in duplicate. Long-chain
cellular fatty acids were extracted and analysed by GC
(MIDI Sherlock) as described by Pot et al. (1994). End
products of glucose metabolism were determined by GLC.
16S rRNA gene fragments were generated by PCR using
universal primers and the amplified products were purified
by using a QIAquick PCR purification kit and sequenced
directly using primers to conserved regions of the 16S
rRNA gene. Sequencing was performed using a PRISM Taq
Dye-deoxy Terminator Cycle Sequencing Kit (Applied
Biosystems) and an automatic DNA sequencer (model
373A; Applied Biosystems). The closest known relatives of
the new isolates were determined by performing database
searches using the FASTA3 search program (www.ebi.ac.uk/
fasta33/nucleotide.html; Pearson & Lipman, 1988). These
sequences and those of other known related strains were
retrieved from GenBank and aligned with the newly
determined sequences using the program SEQTOOLS
(www.seqtools.dk). The resulting multiple sequence alignment was corrected manually using the program GENEDOC
(Nicholas et al., 1997) and a phylogenetic tree was
constructed according to the neighbour-joining method
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89
P. A. Lawson, E. Moore and E. Falsen
(Saitou & Nei, 1987) with the programs SEQTOOLS and
TREEVIEW (Page, 1996). The stability of the groupings was
estimated by bootstrap analysis (1000 replications) using
the same programs.
The two isolates originating from human amniotic fluid
were anaerobic, non-spore-forming, non-motile, Gramnegative rods. Typical cells were 0.8–3.060.5–1.5 mm.
Colonies on blood agar plates after 48 h of incubation at
37 uC under anaerobic conditions were white, smooth,
glistening with an entire edge and less than 1 mm in
diameter. Cells were still viable after 6 h when left in air,
and the catalase reaction was negative for the same period.
No growth was observed in CO2. Using the API Rapid
ID32AN test system the unknown clinical isolates were
positive for N-acetyl-b-D-glucosaminidase, alkaline phosphatase, alanine arylamidase, b-galactosidase, phospho-6b-galactosidase, a-glucosidase, glutamyl-glutamic acid
arylamidase and leucyl-glycine arylamidase. All other
reactions in the API Rapid ID32AN test system were
negative. Employing the API ZYM test kit, positive
reactions were obtained for N-acetyl-b-D-glucosaminidase,
N-AS-BI-phosphohydrolase, alkaline phosphatase, acid
phosphatase, a-glucosidase and b-glucosidase. All other
tests were negative using the API ZYM gallery. Indole was
not produced and nitrate was not reduced. The major end
products of glucose metabolism were acetic and succinic
acids, with minor amounts of isovaleric, isocaproic and
lactic acids also detected. The quantitative fatty acid profile
of CCUG 53648T consisted of C13 : 1 (0.4 %), C14 : 0 (0.9 %),
iso-C14 : 0 (5.6 %), C15 : 0 (0.3 %), iso-C15 : 0 (16.4 %),
anteiso-C15 : 0 (32.3 %), iso-3-OH-C15 : 0 (0.9 %), C16 : 0
(6.4 %), iso-C16 : 0 (9.7 %), 3OH-C16 : 0 (2.0 %), iso-C17 : 0
(3.4 %), anteiso-C17 : 0 (2.9 %), iso-3-OH-C17 : 0 (10.3 %),
2-OH-C17 : 0 (1.1 %), C18 : 0 (1.8 %) and C18 : 1v9c (2.9 %).
To determine the phylogenetic affinities between the
isolates and to other species, the 16S rRNA gene sequences
were amplified by PCR and sequenced. The two isolates
CCUG 53648T and CCUG 43050 were found to be
genetically highly related to each other, displaying 99.5 %
16S rRNA gene sequence similarity. Treeing analysis
revealed the isolates were members of the BacteroidesPrevotella-Porphyromonas rRNA supercluster of organisms
(data not shown), demonstrating a specific affinity with
members of the genus Prevotella. The phylogenetic position
of CCUG 53648T using a reduced dataset is shown in Fig. 1.
Treeing analysis clearly showed that this unknown
bacterium represents a new subline within the genus
Prevotella, sharing a branching node with Prevotella bivia
(95.3 % 16S rRNA gene sequence similarity) supported by
a 100 % bootstrap value. These two species were peripheral
to a subcluster of organisms that included Prevotella
disiens, Prevotella intermedia, Prevotella nigrescens and
Fig. 1. Unrooted tree showing the phylogenetic inter-relationships of Prevotella amnii sp. nov. amongst its nearest relatives.
The tree was constructed using the neighbour-joining method and is based on a comparison of 1330 nt. Bootstrap values, each
expressed as a percentage of 1000 replications, are given at branching points. Bar, 1% sequence divergence.
90
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Prevotella amnii sp. nov.
Prevotella pallens, exhibiting lower levels of sequence
similarity. There is no precise correlation between
percentage 16S rRNA sequence divergence and species
delineation, but it is generally recognized that divergence
values of 3 % or more are significant (Stackebrandt &
Goebel, 1994). Support for the distinctiveness of the
unknown bacterium isolated from amniotic fluid was also
very evident from phenotypic analyses. Tests which are
useful in distinguishing Prevotella amnii from some other
related Prevotella species are shown in Table 1. Our analysis
demonstrated that the closest phylogenetic relative was
Prevotella bivia, an organism originally isolated from the
endometrium and frequently recovered from patients with
pelvic inflammatory diseases (Brook & Frazier, 1997;
Puapermpoonsiri et al., 1997), including its recovery from
amniotic fluid with preterm premature rupture of the
associated membranes (Mikamo et al., 1999). In addition,
sequence database searches revealed that a number of
studies using culture-independent 16S rRNA gene sequencing strategies investigating the vaginal microflora of
healthy women isolated a number of clonal sequences
with almost 100 % similarity to our two isolates. It is
therefore likely that the organism reported here is present
in healthy women, but in certain circumstances can be
enriched for and involved in certain disease processes and
should be regarded as an emerging opportunistic pathogen.
Furthermore, it is likely that some isolates formally
associated with pelvic diseases and presumptively identified
as Prevotella bivia were in fact strains of the novel organism
reported in this article. In particular, the unknown
bacterium can be readily distinguished from Prevotella
bivia by the production of phospho-6-b-galactosidase and
its inability to produce a-fucosidase and glutamyl-glutamic
Table 1. Biochemical characteristics useful in differentiating
Prevotella amnii sp. nov. from type strains of some other
closely related members of the genus Prevotella
Taxa: 1, Prevotella amnii CCUG 53048T; 2, Prevotella bivia CCUG
9557T; 3, Prevotella disiens CCUG 9558T; 4, Prevotella intermedia
CCUG 24041T; 5, Prevotella nigrescens CCUG 9560T; 6, Prevotella
pallens CCUG 39484T. All data are taken from Rapid ID32AN tests
performed at CCUG (www.ccug.se). +, Positive; 2, negative; W,
weak.
acid arylamidase. We therefore consider the formal
description of this novel species and the biochemical
criteria will aid its identification and will facilitate its
recognition in the routine laboratory to distinguish it
from Prevotella bivia and thereby permit the recovery of
additional strains. Furthermore, for unequivocal identification, 16S rRNA gene sequence analysis is recommended
and is now being increasingly incorporated into the clinical
laboratory setting. Based on the presented phenotypic
and phylogenetic evidence, we consider the two unidentified isolates recovered from human amniotic fluid
be assigned to the genus Prevotella, as Prevotella amnii
sp. nov.
Description of Prevotella amnii sp. nov.
Prevotella amnii (am9ni.i. Gr. n. amnion, inner membrane
surrounding the fetus; N.L. gen. n. amnii, of the amnion,
pertaining to the amniotic fluid from which the organism
was first isolated).
Cells consist of Gram-negative rods that are anaerobic,
non-motile and non-spore-forming. Typical cells are 0.8–
3.060.5–1.5 mm. Colonies on blood agar plates after 48 h
of incubation at 37 uC under anaerobic conditions are
white, smooth, glistening with an entire edge and less then
1 mm in diameter. Cells are still viable after 6 h when left
in air. Using the API Rapid ID32AN test system, positive
reactions are produced with N-acetyl-b-D-glucosaminidase,
alkaline phosphatase, alanine arylamidase, phospho-6-bgalactosidase, b-galactosidase, a-glucosidase, glutamyl-glutamic acid arylamidase and leucyl glycine arylamidase.
Using the API ZYM test kit, positive reactions are obtained
for N-acetyl-b-D-glucosaminidase, N-AS-BI-phosphohydrolase, alkaline phosphatase, acid phosphatase, a-glucosidase and b-glucosidase. Indole is not produced and nitrate
is not reduced. Aesculin is hydrolysed but gelatin is not.
Glucose, lactose and maltose are fermented, but cellobiose,
fructose, inositol, mannitol, melibiose, rhamnose, salicin,
sucrose and trehalose are not. The major end products of
glucose metabolism are acetic and succinic acids. The major
long-chain cellular fatty acids consist of iso-C14 : 0, iso-C15 : 0,
anteiso-C15 : 0, C16 : 0, iso-C16 : 0 and iso-3OH-C17 : 0.
The type strain, CCUG 53648T (5JCM 14753T), was
isolated from human amniotic fluid.
Acknowledgements
Characteristic
1
2
3
4
5
6
N-Acetyl-b-D-glucosaminidase
Arginine arylamidase
a-Fucosidase
Phospho-6-b-galactosidase
b-Galactosidase
Glutamyl-glutamic acid
arylamidase
Indole
Raffinose
+
2
2
+
+
2
+
2
+
2
+
+
2
+
2
2
2
2
2
+
+
2
2
2
2
2
+
+
2
2
2
2
2
2
2
+
2
2
2
W
+
2
2
2
W
2
+
+
We thank J. Euzéby for the naming of the novel species, Sofia
Wernersson for DNA sequencing, and Elisabeth Inganäs and Maria
Ohlén for excellent technical assistance. We also thank the depositors
of the strain to the CCUG, Inger Mattsby-Baltzer (Department of
Clinical Bacteriology, University of Göteborg) and S. Kawash (PHLS,
Eastern Hospital, Göteborg).
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